Project Summary Head and neck squamous cell carcinoma (HNSCC) is an invasive malignancy of the upper aerodigestive tract mucosa, accounting for >90% of cancers that arise in the head and neck. HNSCC is the 6th most common cancer by incidence worldwide, with >40,000 new cases in the US, and >500,000 worldwide, each year. The 10 year survival rate is ~50%. Major HNSCC risk factors include tobacco and alcohol use, and infection with Human Papillomavirus (HPV) 16. The limited efficacy of therapies to date is likely due to the genetic heterogeneity of HNSCC, coupled with gaps in knowledge regarding the key driver events and signaling pathways that contribute to the pathogenesis of this neoplasm. We and others have recently elucidated the mutational profile of 412 HNSCC tumors through whole exome sequencing. Our preliminary results demonstrate that the Phosphoinositol-3-Kinase (PI3K) pathway is the most commonly mutated oncogenic pathway in HNSCC, ~41% of tumors in the largest cohort to date have mutations in this pathway. Mutations in PI3K, especially in the most commonly mutated gene in this pathway, PIK3CA, are known to contribute to cancer in HNSCC and other malignancies; though the mechanisms are poorly elucidated. Using a variety of HNSCC screening platforms to identify oncogenic phenotypes, we are defining which PIK3CA mutations can drive cancer in HNSCC. Reverse Phase Protein Arrays (RPPA) will be employed to characterize the cell signaling pathways that these mutations alter to generate cancerous phenotypes in both in vitro and in vivo models of HNSCC harboring PIK3CA mutations, and/or other PI3K alterations such as PTEN loss. Furthermore, we will test targeted PI3K inhibitors in these models, as we anticipate that PI3K alterations will increase the efficacy of these drugs. We will also use RPPA to analyze the ways in which these drugs alter cell signaling pathways in these cancers. Obtaining and comparing these paired datasets will allow me to identify the signaling pathways altered by oncogenic PIK3CA mutations, and determine the signaling pathways that are abrogated by targeted inhibitors, offering evidence-based targets for combined therapy. Finally, an epidemic of HPV(+)HNSCC is emerging, with incidence rates increasing 225% between 1988 and 2004. Retrospective sequencing studies suggest PIK3CA mutations are especially enriched in HPV(+)HNSCC. We will conduct a prospective sequencing project to define the mutational status of PIK3CA and other cancer associated genes in modern clinical HPV(+)HNSCC populations, and initiate experiments to investigate the mechanisms underlying this correlation. The ultimate goal of these studies is to inform and improve the application of targeted next generation therapeutics in HNSCC, in accordance with the mission statement of the NCI.